Project Summary Candida commonly adhere to medical devices, flourishing as a biofilm. A clinical hallmark of biofilm infection is profound antifungal resistance. As effective therapies are not available for these life-threatening infections, understanding how the cells survive existing drug therapies is desperately needed. The extracellular matrix, a distinguishing feature of biofilms, has been linked to drug resistance. This proposed investigation builds logically upon progress in the last funding period, including; the discoveries that 1] key matrix components are delivered via biofilm distinct extracellular vesicle (EVs) as cargo components, 2] exogenous EV add-back assays allow delineation of vesicle cargo constituent roles, 3] ESCRT (endosomal sorting complexes required for transport) pathway components partially impact biofilm EV production and cargo, and 4] a novel antifungal, turbinmicin, inhibits biofilm EV production and matrix production, rendering the cellular community susceptible to antifungals. 5] We further identified a few genetic components necessary for production of these EV and biofilm roles for select vesicle cargo components. However, the functions of the majority of vesicle cargo constituents and the regulatory components of these biofilm pathways remain undefined. Our recent progress helps to solve this mystery. We recently discovered TF mutants with three distinct EV and biofilm resistance and matrix phenotypes. We hypothesize that investigation of these loss of function TFs mutants and their effectors will 1] identify the regulatory network for biofilm EV production and cargo packaging of EVs and 2] uncover roles for EV cargo in biofilm biology, including drug-resistance. Our hypothesis is based upon five main findings. First, we identified a group of TF mutants exhibiting both antifungal susceptibility and extracellular matrix defects, consistent with a matrix protection resistance mechanism. Second, we discovered a subset of drug-susceptible mutants displaying a profound reduction in biofilm EV production. Importantly, the matrix resistance phenotype of these TF mutants is restored via administration of WT EVs. Third, another group of drug-susceptible TF mutants exhibit changes in vesicle cargo but preserved vesicle production. The drug-susceptible phenotype is also reversed for these mutants by addition of WT EVs. Fourth, we find a group of TF mutants that produced elevated quantities of biofilm extracellular vesicles. And fifth¸ exposure of biofilms to turbinmicin reduces not only EV quantity but EV cargo constituents. Our immediate objectives are 1] to define the Candida regulatory pathways that govern vesicle delivery and maturation of the matrix-resistance mechanism and 2] to discern the genetic effectors responsible for production and delivery of these virulence constituents. We seek to test our EV biofilm hypotheses utilizing these three distinct functional TF mutant groups, turbinmicin as a novel pharmacologic...